CN102816695A - Micro-fluidic chip and method for studying effect of fluid shearing force on cell with the micro-fluidic chip - Google Patents
Micro-fluidic chip and method for studying effect of fluid shearing force on cell with the micro-fluidic chip Download PDFInfo
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- CN102816695A CN102816695A CN2011101519055A CN201110151905A CN102816695A CN 102816695 A CN102816695 A CN 102816695A CN 2011101519055 A CN2011101519055 A CN 2011101519055A CN 201110151905 A CN201110151905 A CN 201110151905A CN 102816695 A CN102816695 A CN 102816695A
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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Abstract
The invention provides a micro-fluidic chip and a method for studying an effect of fluid shearing force on a cell with the micro-fluidic chip. The micro-fluidic chip is composed of four cell culture inserts and four fluid passages, wherein the four cell culture inserts have the same size and are parallel mutually; a cell culture insert a and a cell culture insert b share one cell injection port and one cell waste reservoir; a cell culture insert c and a cell culture insert d share one cell injection port and one cell waste reservoir; the upper ends of the four cell culture inserts are separately connected with the four fluid passages; the lower ends of the four cell culture inserts are connected with the same waste reservoir; and the four fluid passages collectively start from a nutrient solution injection port, with the tail ends being connected with the four cell culture inserts respectively. The micro-fluidic chip can be used for studying the effect of the fluid shearing force on the cell.
Description
Technical field
The present invention relates to the micro-fluidic chip technology is applied to the field of biomedical research, the method for a kind of micro-fluidic chip and the effect of research hydrodynamic shear pair cell thereof is provided especially.
Background technology
Liquid-flow is that osseous tissue is discovered mechanical stimulation and aitiogenic crucial regulatory factor; Under external force; Liquid flows to low strain regions from high strain regions, and the various types of cells in the osseous tissue is exposed among the shearing force that causes owing to liquid-flow constantly and a series of reaction takes place.The device of in vitro study hydrodynamic shear pair cell effect is the deceleration loading device according to the rheology principle development; Its ultimate principle is to utilize the mobile nutrient solution that the cell that is attached on the culture medium is produced shearing force; The parallel flat flow chamber is a kind of hydrodynamic shear experiment deceleration loading device commonly used both at home and abroad at present, generates the hydrodynamic shear that multiple proportions changes but be difficult in same parallel flat flow chamber.The micro-fluidic chip technology that developed recently gets up can form the microchannel network on several square centimeters chip, run through total system with controlled fluid, in order to replace a kind of new technology of the breadboard various functions of conventional biological or chemical.We are the cell culture apparatus of technology platform external structure loading hydrodynamic shear with the micro-fluidic chip, for the effect of studying the hydrodynamic shear pair cell provides an effective instrument.
Summary of the invention
The object of the present invention is to provide the method for a kind of micro-fluidic chip and the effect of research hydrodynamic shear pair cell thereof, to be implemented in the effect of in vitro study hydrodynamic shear pair cell.
The invention provides a kind of micro-fluidic chip, it is characterized in that: described micro-fluidic chip mainly is made up of cell cultures pond a 1, cell cultures pond b 2, cell cultures pond c 3, cell cultures pond d 4, fluid channel a 5, fluid channel b 6, fluid channel c 7, fluid channel d 8, nutrient solution injection port 9 and waste liquid pool 10;
---said fluid channel a 5, fluid channel b 6, fluid channel c 7 and fluid channel d 8 originate in nutrient solution injection port 9 jointly; And the end of above-mentioned four fluid channels respectively with cell cultures pond a 1, cell cultures pond b 2, cell cultures pond c 3, cell cultures pond d 4 corresponding successively connections, and cell cultures pond a 1, cell cultures pond b 2, cell cultures pond c 3 and cell cultures pond d 4 link to each other with same waste liquid pool 10.
Wherein, Said cell cultures pond a 1, cell cultures pond b 2, cell cultures pond c 3, cell cultures pond d 4 equal and opposite in directions also are parallel to each other; Cell cultures pond a 1 and cell cultures pond b 2 a shared cell injection port a 11 and cell waste liquid pool a 13, cell cultures pond c 3 and cell cultures pond b 4 a shared cell injection port b 12 and cell waste liquid pool b 14; The height of fluid channel a 5, fluid channel b 6, fluid channel c 7, fluid channel d 8 is consistent, and the width of fluid channel a 5, fluid channel b 6, fluid channel c 7, fluid channel d 8 is different with length; Preferably; The height of fluid channel a 5, fluid channel b 6, fluid channel c 7, fluid channel d 8 is 100 μ m; The width of fluid channel a 5, fluid channel b 6, fluid channel c 7, fluid channel d 8 is respectively 400 μ m, 90.8 μ m, 51.9 μ m, 49.2 μ m, and the length of fluid channel a 5, fluid channel b 6, fluid channel c 7, fluid channel d 8 is respectively 4.85 μ m, 12.74 μ m, 29.88 μ m, 138.27 μ m.
A kind of micro-fluidic chip provided by the invention; The flow field that liquid flow produces in cell cultures pond a 1, cell cultures pond b 2, cell cultures pond c 3, cell cultures pond d 4 behind described fluid channel a 5, fluid channel b 6, fluid channel c 7, fluid channel d 8 is stable; Specifically see Fig. 4; And the hydrodynamic shear that liquid flow produces in cell cultures pond a 1, cell cultures pond b 2, cell cultures pond c 3, cell cultures pond d 4 behind said fluid channel a 5, fluid channel b 6, fluid channel c 7, fluid channel d 8 is that multiple proportions changes; The ratio of the hydrodynamic shear that in cell cultures pond a 1, cell cultures pond b 2, cell cultures pond c 3, cell cultures pond d 4, produces is 1:5:25:125, specifically sees Fig. 5.
The upper layer of material of micro-fluidic chip is the PDMS polymkeric substance among the present invention, with the irreversible sealing-in of the glass material of lower floor, and makes the PDMS surface be converted into hydrophily by hydrophobic state after the plasma treatment.
Use the method for micro-fluidic chip research hydrodynamic shear pair cell effect among the present invention, concrete process is following:
---through the cell injection port with cell inoculation in four cell cultures ponds;
---chip placed 37 ℃ CO
2Cultivated 12 ~ 24 hours in the incubator, make cell fully adherent, the cell in each cell cultures pond is counted;
---micro-injection pump is connected with the nutrient solution injection port, and pair cell carries out continous perfusion and cultivates;
---chip placed 37 ℃ CO
2Cultivated 24 ~ 72 hours in the incubator, investigate the effect of hydrodynamic shear pair cell.
Micro-fluidic chip provided by the invention; Its advantage is: can be on more than one square centimeters chip culturing cell; And pair cell applies the hydrodynamic shear that multiple proportions changes, and investigates the influence that hydrodynamic shear changes pair cell, has important biomedical researching value and economic worth.
Description of drawings
Fig. 1 shows the structural representation of micro-fluidic chip, wherein (1) cell cultures pond a, (2) cell cultures pond b, (3) cell cultures pond c, (4) cell cultures pond d, (5) fluid channel a, (6) fluid channel b, (7) fluid channel c, (8) fluid channel d, (9) nutrient solution injection port, (10) waste liquid pool, (11) cell injection port a, (12) cell injection port b, (13) cell waste liquid pool a, (14) cell waste liquid pool b;
Fig. 2 shows micro-fluidic chip photo in kind, and wherein (15) are that PDMS top layer, (16) are substrate of glass;
Fig. 3 shows the synoptic diagram of the microscopically of four fluid channels;
Flow Field Distribution mimic diagram in Fig. 4 showed cell cultivation pool;
Fig. 5 shows the size of the different cell cultures pond inner fluid shearing force that calculates;
Fig. 6 perfusion is cultivated and is carried out Rh123-Hoechst dyeing after 48 hours; The upgrowth situation figure that shows MC3T3-E1 cell in the different cell cultures pond, wherein (1) cell cultures pond a, (2) cell cultures pond b, (3) cell cultures pond c, (4) cell cultures pond d;
Fig. 7 shows that perfusion cultivates after 48 hours MC3T3-E1 cell proliferation index variation, wherein (1) cell cultures pond a, (2) cell cultures pond b, (3) cell cultures pond c, (4) cell cultures pond d in the different cell cultures pond.
Embodiment
Following embodiment will further explain the present invention, but therefore not limit the present invention.
A kind of micro-fluidic chip; Concrete structure is as shown in Figure 1; Its pictorial diagram is as shown in Figure 2; Its chip upper layer of material is the PDMS polymkeric substance, is packaged on lower floor's glass surface through irreversible sealing technology, mainly is made up of cell cultures pond a 1, cell cultures pond b 2, cell cultures pond c 3, cell cultures pond d 4 and fluid channel a 5, fluid channel b 6, fluid channel c 7, fluid channel d 8, nutrient solution injection port 9 and waste liquid pool 10; Said fluid channel a 5, fluid channel b 6, fluid channel c 7 and fluid channel d 8 originate in nutrient solution injection port 9 jointly; And the end of above-mentioned four fluid channels respectively with cell cultures pond a 1, cell cultures pond b 2, cell cultures pond c 3, cell cultures pond d 4 corresponding successively connections, and cell cultures pond a 1, cell cultures pond b 2, cell cultures pond c 3 and cell cultures pond d 4 link to each other with same waste liquid pool 10; The height of described fluid channel a 5, fluid channel b 6, fluid channel c 7, fluid channel d 8 is 100 μ m; The width of fluid channel a 5, fluid channel b 6, fluid channel c 7, fluid channel d 8 is respectively 400 μ m, 90.8 μ m, 51.9 μ m, 49.2 μ m, and the length of fluid channel a 5, fluid channel b 6, fluid channel c 7, fluid channel d 8 is respectively 4.85 μ m, 12.74 μ m, 29.88 μ m, 138.27 μ m.
Use the micro-fluidic chip among the embodiment 1; Micro-injection pump is connected with the nutrient solution injection port; Carry out the perfusion of nutrient solution with 0.08 μ l/ minute flow velocity; Measure the Flow Field Distribution in the above-mentioned micro-fluidic chip cell cultures pond, wherein the Flow Field Distribution in four cell cultures ponds (1) cell cultures pond a, (2) cell cultures pond b, (3) cell cultures pond c, (4) the cell cultures pond d is even, specifically sees Fig. 4.
Use the micro-fluidic chip among the embodiment 1; Micro-injection pump is connected with the nutrient solution injection port; Carry out the perfusion of nutrient solution with 0.08 μ l/ minute flow velocity; Measure the size of the different cell cultures of above-mentioned micro-fluidic chip pond inner fluid shearing force, wherein (1) cell cultures pond a, (2) cell cultures pond b, (3) cell cultures pond c, (4) cell cultures pond d, the hydrodynamic shear among a of cell cultures pond is far longer than the hydrodynamic shear among cell cultures pond b, c, the d; Their ratio is about 1:5:25:125, specifically sees Fig. 5.
Use the micro-fluidic chip among the embodiment 1 to investigate the influence that hydrodynamic shear changes on cell proliferation, through the cell injection port with preosteoblast be the MC3T3-E1 cell inoculation in four cell cultures ponds, chip placed 37 ℃ CO
2Cultivated 24 hours in the incubator, make cell fully adherent, the cell in each cell cultures pond is counted.Micro-injection pump is connected with the nutrient solution injection port; Carry out perfusion with 0.08 μ l/ minute flow velocity pair cell and cultivate, pair cell carries out Rh123-Hoechst dyeing after 48 hours, investigates the upgrowth situation of cell; See Fig. 6, and the cell in each cell cultures pond is counted.Variation through cell quantity before and after relatively perfusion is cultivated calculates the cell proliferation index in each cell cultures pond, sees Fig. 7.Hydrodynamic shear among the d of cell cultures pond is maximum; Cell proliferation rate in it is the slowest; Compare with the cell proliferation index in the b with cell cultures pond a, significant difference is arranged, therefore; Under the hydrodynamic shear effect, the ability of cell proliferation among the d of cell cultures pond is compared remarkable decline with cell cultures pond a with the cell in the b.
Claims (8)
1. micro-fluidic chip, it is characterized in that: described micro-fluidic chip mainly is made up of cell cultures pond a (1), cell cultures pond b (2), cell cultures pond c (3), cell cultures pond d (4), fluid channel a (5), fluid channel b (6), fluid channel c (7), fluid channel d (8), nutrient solution injection port (9) and waste liquid pool (10);
---said fluid channel a (5), fluid channel b (6), fluid channel c (7) and fluid channel d (8) originate in nutrient solution injection port (9) jointly; And the end of above-mentioned four fluid channels respectively with cell cultures pond a (1), cell cultures pond b (2), cell cultures pond c (3), the corresponding successively connection of cell cultures pond d (4), and cell cultures pond a (1), cell cultures pond b (2), cell cultures pond c (3) and cell cultures pond d (4) link to each other with same waste liquid pool (10).
2. according to the described micro-fluidic chip of claim 1; It is characterized in that: said cell cultures pond a (1), cell cultures pond b (2), cell cultures pond c (3), cell cultures pond d (4) equal and opposite in direction also are parallel to each other; Cell cultures pond a (1) and the shared cell injection port a of cell cultures pond b (2) (11) and a cell waste liquid pool a (13), cell cultures pond c (3) and the shared cell injection port b of cell cultures pond b (4) (12) and a cell waste liquid pool b (14).
3. according to the described micro-fluidic chip of claim 1; It is characterized in that: the height of described fluid channel a (5), fluid channel b (6), fluid channel c (7), fluid channel d (8) is identical, and the width of fluid channel a (5), fluid channel b (6), fluid channel c (7), fluid channel d (8) is different with length.
4. according to the described micro-fluidic chip of claim 3; It is characterized in that: liquid flow is through described fluid channel a (5), fluid channel b (6), fluid channel c (7), fluid channel d (8), and the flow field that in cell cultures pond a (1), cell cultures pond b (2), cell cultures pond c (3), cell cultures pond d (4), produces is stable.
5. according to the described micro-fluidic chip of claim 3; It is characterized in that: liquid flow is through said fluid channel a (5), fluid channel b (6), fluid channel c (7), fluid channel d (8); The hydrodynamic shear that in cell cultures pond a (1), cell cultures pond b (2), cell cultures pond c (3), cell cultures pond d (4), produces is that multiple proportions changes, and the ratio of the hydrodynamic shear that in cell cultures pond a (1), cell cultures pond b (2), cell cultures pond c (3), cell cultures pond d (4), produces is 1:5:25:125.
6. according to the described micro-fluidic chip of claim 1; It is characterized in that: the height of described fluid channel a (5), fluid channel b (6), fluid channel c (7), fluid channel d (8) is 100 μ m; The width of fluid channel a (5), fluid channel b (6), fluid channel c (7), fluid channel d (8) is respectively 400 μ m, 90.8 μ m, 51.9 μ m, 49.2 μ m, and the length of fluid channel a (5), fluid channel b (6), fluid channel c (7), fluid channel d (8) is respectively 4.85 μ m, 12.74 μ m, 29.88 μ m, 138.27 μ m.
7. according to the described micro-fluidic chip of claim 1, it is characterized in that: the upper layer of material of said micro-fluidic chip is the PDMS polymkeric substance, and lower floor is a glass.
8. method according to the effect of the described micro-fluidic chip of claim 1 research hydrodynamic shear pair cell, detailed process is following:
---through the cell injection port with cell inoculation in four cell cultures ponds;
---chip placed 37 ℃ CO
2Interior 12 ~ 24 hours of incubator makes cell fully adherent, and the cell in each cell cultures pond is counted;
---micro-injection pump is connected with the nutrient solution injection port, and pair cell carries out continous perfusion and cultivates;
---chip placed 37 ℃ CO
2Cultivated 24 ~ 72 hours in the incubator, investigate the effect of hydrodynamic shear pair cell.
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Cited By (5)
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| CN104293666A (en) * | 2014-09-11 | 2015-01-21 | 大连理工大学 | Micro-fluidic chip device for detecting interaction between two different unicells |
| CN106119079A (en) * | 2016-07-06 | 2016-11-16 | 四川大学 | Clearance flow cyto-mechanics biology experimental installation between one |
| CN108018207A (en) * | 2016-10-28 | 2018-05-11 | 华东理工大学 | The biomechanical system with stretching culture is sheared for cell flow |
| CN108339580A (en) * | 2018-03-20 | 2018-07-31 | 哈尔滨工业大学深圳研究生院 | A kind of fluid shearing force generating mechanism and hydrodynamic shear generation method |
| CN112300930A (en) * | 2019-07-31 | 2021-02-02 | 上海新微技术研发中心有限公司 | Microfluidic experimental plate and double-sided cell culture method |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293666A (en) * | 2014-09-11 | 2015-01-21 | 大连理工大学 | Micro-fluidic chip device for detecting interaction between two different unicells |
| CN104293666B (en) * | 2014-09-11 | 2016-06-22 | 大连理工大学 | The micro flow control chip device of the interphase interaction that two kinds of differences are unicellular |
| CN106119079A (en) * | 2016-07-06 | 2016-11-16 | 四川大学 | Clearance flow cyto-mechanics biology experimental installation between one |
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| CN108339580A (en) * | 2018-03-20 | 2018-07-31 | 哈尔滨工业大学深圳研究生院 | A kind of fluid shearing force generating mechanism and hydrodynamic shear generation method |
| WO2019178923A1 (en) * | 2018-03-20 | 2019-09-26 | 哈尔滨工业大学深圳研究生院 | Fluid shear stress generation device and fluid shear stress generation method |
| CN108339580B (en) * | 2018-03-20 | 2020-01-14 | 哈尔滨工业大学深圳研究生院 | Fluid shear force generation device and fluid shear force generation method |
| CN112300930A (en) * | 2019-07-31 | 2021-02-02 | 上海新微技术研发中心有限公司 | Microfluidic experimental plate and double-sided cell culture method |
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